Quantitative high-throughput assay to measure MC4R-induced intracellular calcium.

The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.

Metallomics Analysis for Assessment of Toxic Metal Burdens in Infants/Children and Their Mothers: Early Assessment and Intervention Are Essential.

Accumulation of toxic metals in infants/children is of serious concern worldwide, from the viewpoint of their harmful effects on the normal growth and development. This metallomics study investigates the extent of toxic metal burdens in infants/children and the relationship to those in their mothers for 77 child/mother pair subjects. For mercury, its geometric mean concentration in infants/children was of similar level to that in their mothers, and a high-significant close correlation was observed between infants/children and their mothers (ß = 0.758, r = 0.539, p < 0.0001). A significant but less intimate mother/child relationship was observed for arsenic (ß = 0.301, r = 0.433), lead (ß = 0.444, r = 0.471) and aluminum (ß = 0.379, r = 0.451). Remarkably, the burden levels of lead, cadmium and aluminum in infants/children were approximately three times higher than those in their mothers (p < 0.0001), and the burden levels in some individuals were several tens of times higher than in the mothers. In contrast, some essential metal levels such as zinc, magnesium and calcium in infants/children were significantly lower than those in their mothers, and 29 individuals (37.7%) in the child subjects were estimated to be zinc-deficient. In addition, significant inverse correlations were observed between zinc and lead (r = -0.267, p = 0.019), and magnesium and arsenic (r = -0.514, p < 0.0001). These findings suggest that these toxic metal burdens and essential metal deficiencies in infants/children are of serious concern for their neurodevelopment, indicating that the early assessment and intervention are crucial. It is expected that larger epidemiological and intervention studies will provide a reasonable and essential pathway for intervention of neurodevelopment disorders.

Biotechnological Applications of Paenibacillus sp. D9 Lipopeptide Biosurfactant Produced in Low-cost Substrates.

The present study assesses the Paenibacillus sp. D9 lipopeptide biosurfactant synthesis in cheap substrates including functional properties and applicability for varying biotechnological processes. Different experimental setups were made for oil dispersion, heavy metals removals from contaminated environments, and washing performance. The study revealed surface tension activities of 31.7-32.7 mN/m, and maximum biosurfactant yield of more than 8 g/L. Removals of 85.90%, 98.68%, 99.97%, 63.28%, 99.93%, and 94.22% were obtained for Ca, Cu, Fe, Mg, Ni, and Zn, respectively from acid mine effluents. In comparison with chemical surfactants, there was pronounced removal of heavy metals from wastewater, contaminated sands, and vegetable matter, as well as improved oil dispersing activity. A comparative study revealed that biosurfactant was more efficient (> 60%) for removal of tomato sauce and coffee stains than chemical surfactants (< 50%). Thus, lipopeptide biosurfactants are green biomolecules reducing hazards and contaminations within the environment. The future use of this lipopeptide biosurfactant is greatly promising in biotechnology.

Hydrochars produced with by-products from the sucroenergetic industry: a study of extractor solutions on nutrient and organic carbon release.

Hydrothermal carbonization transforms biomass into value-added material called hydrochar. The release of nutrients (P, N, Ca, Mg, and K) and organic carbon (TOC) from hydrochar in different extractive solutions was investigated in this study. Two sets of hydrochar were produced: (i) hydrochar prepared from sugarcane bagasse and vinasse mixture (BV-HC) and (ii) hydrochar prepared by the addition of H3PO4 to this mixture (BVA-HC). Both hydrochar types released significative amounts of nutrient and organic carbon, mainly Ca (5.0 mg g-1) in the mixture (KCl, K2SO4, NaOH, 1:1:1) extractive solution and TOC (72.6 mg g-1) in the NaOH extractive solution, for BV-HC. Nutrient release was influenced by pH and ionic strength. The release of P, Ca, and Mg was affected by the presence of insoluble phosphate phases in BVA-HC. The release of nutrients P, N, Ca, Mg, and K and organic carbon demonstrated that hydrochar has potential for soil application purposes.

New Sources of Calcium (Chicken Eggshells, Chelates) - Preparation of Raw Material and Tablets.

BACKGROUND: There are many calcium supplements available in the market, especially those containing calcium in the form of carbonate, which unfortunately is not absorbed by the body to a sufficient degree. METHOD: Therefore, an attempt was made to prepare new sources of calcium, consuming the chicken eggshells as natural raw materials, which were used in preparation of tablets containing calcium carbonate and calcium citrate as well as tablets with calcium carbonate and calcium bisglycinate. The influence of raw material properties on the pharmaceutical availability of calcium from the obtained tablets was investigated. RESULTS: Based on the obtained calcium release profiles from the prepared tablets, it was found that the optimal source of calcium is a preparation containing calcium from chicken eggshells. It was found that both chicken eggshells and calcium bisglycinate (chelate) may be new, prospective sources of calcium. Calcium citrate prepared using eggshells as starting materials and bisglycinate is completely released within no more than 150 minutes. CONCLUSION: In turn, calcium carbonate added to calcium bisglycinate statistically significantly prolonges the release of calcium ions to 4 hours.

Effect of different methods of Ca2+ extraction from PSII oxygen-evolving complex on the QA- oxidation kinetics.

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn4CaO5 of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca2+ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca2+ extraction from OEC on the kinetics of the reduced primary electron acceptor (QA-) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from QA- to the secondary quinone acceptor QB. Electron transfer from QA- to QB in photosystem II membranes with an occupied QB site was slowed down by a factor of 8. However, addition of EGTA or CaCl2 to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of QA- oxidation by QB in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca2+ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the QA- to QB electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca2+ depletion.

Ca removal and Mg recovery from flue gas desulfurization (FGD) wastewater by selective precipitation.

Selective removal of Ca and recovery of Mg by precipitation from flue gas desulfurization (FGD) wastewater has been investigated. Thermodynamic analysis of four possible additives, Na2CO3, Na2C2O4, NaF and Na2SO4, indicated that both carbonate and oxalate could potentially provide effective separation of Ca via precipitation from Mg in FGD wastewater. However, it was found experimentally that the carbonate system was not as effective as oxalate in this regard. The oxalate system performed considerably better, with Ca removal efficiency of 96% being obtained, with little Mg inclusion at pH 6.0 when the dosage was ×1.4 the stoichiometric requirement. On this basis, the subsequent recovery process for Mg was carried out using NaOH with two-step precipitation. The product was confirmed to be Mg(OH)2 (using X-ray diffraction and thermo gravimetric analysis) with elemental analysis suggesting a purity of 99.3 wt.%.

Development of a High-Throughput Ion-Exchange Resin Characterization Workflow.

A novel high-throughout (HTR) ion-exchange (IEX) resin workflow has been developed for characterizing ion exchange equilibrium of commercial and experimental IEX resins against a range of different applications where water environment differs from site to site. Because of its much higher throughput, design of experiment (DOE) methodology can be easily applied for studying the effects of multiple factors on resin performance. Two case studies will be presented to illustrate the efficacy of the combined HTR workflow and DOE method. In case study one, a series of anion exchange resins have been screened for selective removal of NO3- and NO2- in water environments consisting of multiple other anions, varied pH, and ionic strength. The response surface model (RSM) is developed to statistically correlate the resin performance with the water composition and predict the best resin candidate. In case study two, the same HTR workflow and DOE method have been applied for screening different cation exchange resins in terms of the selective removal of Mg2+, Ca2+, and Ba2+ from high total dissolved salt (TDS) water. A master DOE model including all of the cation exchange resins is created to predict divalent cation removal by different IEX resins under specific conditions, from which the best resin candidates can be identified. The successful adoption of HTR workflow and DOE method for studying the ion exchange of IEX resins can significantly reduce the resources and time to address industry and application needs.

Microalgal bacterial flocs treating paper mill effluent: A sunlight-based approach for removing carbon, nitrogen, phosphorus, and calcium.

Treatment of upflow anaerobic sludge blanket (UASB) effluent from a paper mill in aerated activated sludge reactors involves high aeration costs. Moreover, this calcium-rich effluent leads to problematic scale formation. Therefore, a novel strategy for the aerobic treatment of paper mill UASB effluent in microalgal bacterial floc sequencing batch reactors (MaB-floc SBRs) is proposed, in which oxygen is provided via photosynthesis, and calcium is removed via bio-mineralization. Based on the results of batch experiments in the course of this study, a MaB-floc SBR was operated at an initial neutral pH. This SBR removed 58±21% organic carbon, 27±8% inorganic carbon, 77±5% nitrogen, 73±2% phosphorus, and 27±11% calcium. MaB-flocs contained 10±3% calcium, including biologically-influenced calcite crystals. The removal of calcium and inorganic carbon by MaB-flocs significantly decreased when inhibiting extracellular carbonic anhydrase (CA), an enzyme that catalyses the hydration and dehydration of CO2. This study demonstrates the potential of MaB-floc SBRs for the alternative treatment of calcium-rich paper mill effluent, and highlights the importance of extracellular CA in this treatment process.

Phosphorus removal from aqueous solution using a novel granular material developed from building waste.

In this study, a granular material (GM) developed from building waste was used for phosphate removal from phosphorus-containing wastewater. Batch experiments were executed to investigate the phosphate removal capacity of this material. The mechanism of removal proved to be a chemical precipitation process. The characteristics of the material and resulting precipitates, the kinetics of the precipitation and Ca2+ liberation processes, and the effects of dosage and pH were investigated. The phosphate precipitation and Ca2+ liberation processes were both well described by a pseudo-second-order kinetic model. A maximum precipitation capacity of 0.51 ± 0.06 mg g-1 and a liberation capacity of 6.79 ± 0.77 mg g-1 were measured under the experimental conditions. The processes reached equilibrium in 60 min. The initial solution pH strongly affected phosphate removal under extreme conditions (pH <4 and pH >10). The precipitates comprised hydroxyapatite and brushite. This novel GM can be considered a promising material for phosphate removal from wastewater.

Flow of essential elements in subcellular fractions during oxidative stress.

Essential trace elements are commonly found in altered concentrations in the brains of patients with neurodegenerative diseases. Many studies in trace metal determination and quantification are conducted in tissue, cell culture or whole brain. In the present investigation, we determined by ICP-MS Fe, Cu, Zn, Ca, Se, Co, Cr, Mg, and Mn in organelles (mitochondria, nuclei) and whole motor neuron cell cultured in vitro. We performed experiments using two ways to access oxidative stress: cell treatments with H2O2 or Aß-42 peptide in its oligomeric form. Both treatments caused accumulation of markers of oxidative stress, such as oxidized proteins and lipids, and alteration in DNA. Regarding trace elements, cells treated with H2O2 showed higher levels of Zn and lower levels of Ca in nuclei when compared to control cells with no oxidative treatments. On the other hand, cells treated with Aß-42 peptide in its oligomeric form showed higher levels of Mg, Ca, Fe and Zn in nuclei when compared to control cells. These differences showed that metal flux in cell organelles during an intrinsic external oxidative condition (H2O2 treatment) are different from an intrinsic external neurodegenerative treatment.

[NOTHROMBEL EFFECT ON THE FORMATION OF PLATELET-LEUKOCYTE COMPLEXES INDUCED BY THROMBIN].

The effects of the Nothrombel on the formation of platelet-leukocyte complexes (PLCs) induced by thrombin was studied. It was shown, that Nothrombel dose-dependently inhibited the formation of PLCs. Its activity is higher than the activity of the comparison compounds Aspirin. The half maximal effective concentration (EC50) for Nothrombel is 1.75 mMol/mL, for Aspirin is much more than 2.5 mMol/mL. The inhibition mechanism of the PLCs formation by Nothrombel caused by the ability of this drug to inhibit the P-selectin translocation on the platelet membrane, the expression of membrane complex GPIb-IX-V, the mobilization of cytoplasmic calcium in platelets, as well as, apparently, its inhibitory effect on platelet P2Y12 purine receptors.

[The application of Fluo-3 AM in measurement of level of cytoplasmic calcium in thrombocytes by flow cytofluorometry].

The study was carried out to evaluate possibility of applying technique of thrombin-induced increasing of concentration of Ca in cytoplasm of Fluo-3-colored thrombocytes as an experimental model of studying mechanism of action of anti-thrombocytes medications in vitro. The effect of anti-thrombocyte substances on thrombin-induced increasing of the level of cytoplasmic Ca in thrombocytes was analyzed on example of acetylsalicylic acid. The measurement of concentration of cytoplasmic Ca was implemented using flow cytometry technique with fluorescent probe Fluo-3 AM. It is established that in the given test acetylsalicylic acid inhibits thrombin-induced increasing of cytoplasmic Ca at 0.125-5.0 mk/mol concentrations. This occurrence testifies that in the mechanism of effect of acetylsalicylic acid the suppression of thromboxane path ceases to be a leading one. The proposed methodical approach permits evaluating anti-thrombocite effect of substances according their impact to the level of cytoplasmic Ca in thrombocytes in vitro. However, this approach has a number of limitations preventing wide-spread application of the given technique.

Direct imaging of elemental distributions in tissue sections by laser ablation mass spectrometry.

We present a strategy for imaging of elements in biological tissues using laser ablation (LA) mass spectrometry (MS), which was compared to laser ablation inductively coupled plasma (LA-ICP) MS. Both methods were adopted for quantitative imaging of elements in mouse kidney, as well as traumatic brain injury model tissue sections. MS imaging (MSI) employing LA provides quantitative data by comparing signal abundances of sodium from tissues to those obtained by imaging quantitation calibration standards of the target element applied to adjacent control tissue sections. LA-ICP MSI provided quantitative data for several essential elements in both brain and kidney tissue sections using a dried-droplet approach. Both methods were used to image a rat model of traumatic brain injury, revealing accumulations of sodium and calcium in the impact area and its peripheral regions. LA MSI is shown to be a viable option for quantitative imaging of specific elements in biological tissue sections.

A sulphonated carbon dot-chitosan hybrid hydrogel nanocomposite as an efficient ion-exchange film for Ca2+ and Mg2+ removal.

We have developed a hybrid hydrogel nanocomposite film via conjugation of oxidised carbon dots synthesized from 11-mercaptoundecanoic acid with chitosan. The potential applicability of the film was then successfully tested for the removal of Ca2+ and Mg2+ ions from solution.

Simultaneous nitrogen, phosphorous, and hardness removal from reverse osmosis concentrate by microalgae cultivation.

While reverse osmosis (RO) is a promising technology for wastewater reclamation, RO concentrate (ROC) treatment and disposal are important issues to consider. Conventional chemical and physical treatment methods for ROC present certain limitations, such as relatively low nitrogen and phosphorus removal efficiencies as well as the requirement of an extra process for hardness removal. This study proposes a novel biological approach for simultaneous removal of nitrogen, phosphorus, and calcium (Ca(2+)) and magnesium (Mg(2+)) ions from the ROC of municipal wastewater treatment plants by microalgal cultivation and algal biomass production. Two microalgae strains, Chlorella sp. ZTY4 and Scenedesmus sp. LX1, were used for batch cultivation of 14-16 days. Both strains grew well in ROC with average biomass production of 318.7 mg/L and lipid contents up to 30.6%, and nitrogen and phosphorus could be effectively removed with efficiencies of up to 89.8% and 92.7%, respectively. Approximately 55.9%-83.7% Ca(2+) could be removed from the system using the cultured strains. Mg(2+) removal began when Ca(2+) precipitation ceased, and the removal efficiency of the ion could reach up to 56.0%. The most decisive factor influencing Ca(2+) and Mg(2+) removal was chemical precipitation with increases in pH caused by algal growth. The results of this study provide a new biological approach for removing nitrogen, phosphorous, and hardness from ROC. The results suggest that microalgal cultivation presents new opportunities for applying an algal process to ROC treatment. The proposed approach serves dual purposes of nutrient and hardness reduction and production of lipid rich micro-algal biomass.

A novel bio-electronic tongue using different cellobiose dehydrogenases to resolve mixtures of various sugars and interfering analytes.

A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca(2+) in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direct electron transfer mode. The Artificial Neural Network (ANN) used in the BioET system to interpret the voltammetric data was able to provide a correct prediction of the concentrations of the analytes considered. Correlation coefficients in the comparison of obtained vs. expected concentrations were highly significant, especially for lactose (R(2)=0.975) and Ca(2+) (R(2)=0.945). This BioET application has a high potential especially for the food and dairy industry and also, if further miniaturised in screen printed format, for its in-situ use.

Earliest tea as evidence for one branch of the Silk Road across the Tibetan Plateau.

Phytoliths and biomolecular components extracted from ancient plant remains from Chang'an (Xi'an, the city where the Silk Road begins) and Ngari (Ali) in western Tibet, China, show that the tea was grown 2100 years ago to cater for the drinking habits of the Western Han Dynasty (207BCE-9CE), and then carried toward central Asia by ca.200CE, several hundred years earlier than previously recorded. The earliest physical evidence of tea from both the Chang'an and Ngari regions suggests that a branch of the Silk Road across the Tibetan Plateau, was established by the second to third century CE.

Effects of natural organic matter on calcium and phosphorus co-precipitation.

Phosphorus (P), calcium (Ca) and natural organic matter (NOM) naturally occur in all aquatic ecosystems. However, excessive P loads can cause eutrophic or hyper-eutrophic conditions in these waters. As a result, P regulation is important for these impaired aquatic systems, and Ca-P co-precipitation is a vital mechanism of natural P removal in many alkaline systems, such as the Florida Everglades. The interaction of P, Ca, and NOM is also an important factor in lime softening and corrosion control, both critical processes of drinking water treatment. Determining the role of NOM in Ca-P co-precipitation is important for identifying mechanisms that may limit P removal in both natural and engineered systems. The main goal of this research is to assess the role of NOM in inhibiting Ca and P co-precipitation by: (1) measuring how Ca, NOM, and P concentrations affect NOM's potential inhibition of co-precipitation; (2) determining the effect of pH; and (3) evaluating the precipitated solids. Results showed that Ca-P co-precipitation occurs at pH 9.5 in the presence of high natural organic matter (NOM) (≈30 mg L(-1)). The supersaturation of calcite overcomes the inhibitory effect of NOM seen at lower pH values. Higher initial P concentrations lead to both higher P precipitation rates and densities of P on the calcite surface. The maximum surface density of co-precipitated P on the precipitated calcite surface increases with increasing NOM levels, suggesting that NOM does prevent the co-precipitation of Ca and P.

[Adsorption of calcium ion from aqueous solution using Na(+)-conditioned clinoptilolite for hot-water softening].

This work investigated adsorptive removal of calcium ion (Ca2+) by virtue of Na(+) -conditioned clinoptilolite simulating the process of softening for industrial hot-water system. Influential factors such as the activation/regeneration of sorbent and solution pH were tested. The kinetics/thermodynamics for adsorption of Ca2+ were analyzed and discussed. Results showed that: (1) The adsorption rate was in good agreement with the pseudo-second order kinetic models, and the process of adsorption better followed the Langmuir model; (2) Higher solution temperature allowed an enhanced efficiency on Ca2+ removal, albeit the maximum adsorption capacity of Na(+)-conditioned clinoptilolite was hardly affected; (3) The process of adsorption was dominated by chemisorption, and also characterized by entropy increase with spontaneous/endothermic nature; (4) Solution temperature was suggested to be controlled within the range of 6 to 10, and more than 9 times of sorbent regeneration could be ensured for an effective adsorption towards Ca2+ with initial concentration less than 20 mg x L(-1). It was demonstrated that the activated clinoptilolite should be a promising alternative adsorbent for industrial hot-water softening.